Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase

Ellen C. Minnihan, Mohammad R. Seyedsayamdost, Joanne Stubbe

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Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 50-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y122 ) cofactor which, in the presence of S and E, oxidizes C 439 in α to a thiyl radical, C439, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y122 → W48 → Y356 in β2 to Y731 → Y730 → C439 in α2. 3-Aminotyrosine (NH2Y) has been sitespecifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH2Y) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y 356F-β2:Y731NH2Y-α2, Y 356F-β2:Y730NH2Y-α2, and wt-β2:Y731F/Y730NH2Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH2Y was detected. To study off-pathway oxidation, Y413, located 5Å from Y 730 and Y731 but not implicated in long-range oxidation, was examined. Evidence for NH2Y413 was sought in three complexes: wt-β2:Y413NH2Y-α2 (a), wt-β2:Y731F/Y413NH2Y-α2 (b), and Y356F-β2: Y413NH2Y-α2 (c). With (a), NH2Y was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant forNH2Y formation was 0.2-0.3% of that observed at 731 and 730, and with (c), noNH 2Y was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C439.

Original languageEnglish (US)
Pages (from-to)12125-12132
Number of pages8
Issue number51
StatePublished - Dec 29 2009
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry


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